One human antilymphocyte serum can exhibit many different types of reactivity including cytotoxic anti HLA, anti T, and/or anti B cell activity, blocking in the mixed lymphocyte reaction (MLR) and the ability to mediate the lymphocyte-antibody lymphocytolytic interaction (LALI). The anti HLA activity may be "monospecific" or may cross react with antigens not present on the immunizing cell. Studies on the interrelationships of these reactions using standard immunologic separation techniques are often ambiguous. The purpose of the proposed research is to use isoelectric focusing (IEF) to isolate and characterize the populations of antibodies which mediate anti HLA cytotoxicity, MLR blocking of LALI. IEF is capable of fine discrimination between populations of antibodies having closely related specificities and has previously been used to good advantage in studies of human and lymphocyte reagents. Fractions prepared from selected antisera by isoelectric focusing will be studied for activity in the cytotoxicity test for HLA, T and B cell antigens, and for activity in LALI and blocking in the MLR. The isoelectric point of the different types of activity in LALI and blocking in the MLR. The isoelectric point of the different types of activity will be examined along with the immunoglobulin composition of active fractions and the role of specific immunoglobulin classes and subclasses in the reaction. These experiments should result in a better understanding of the serological nature of the HLA antigens particularly in regard to the role of antibody in cross reactivity and in a clearer definition of the relation of HLA to MLR blocking and LALI.